Compositions and methods for production of exofucosylated cells for clinical applications

Abstract

The present disclosure provides, inter alia, compositions and methods for detecting changes in level of expression of cell-surface Type 2 terminal lactosamines on a population of cultured cells propagated under different conditions. The disclosure also provides compositions and methods for enforcing stably expressed glycans on human cells. In certain embodiments, the compositions and/or methods utilize one or more members of the α(1,3)-fucosyltransferase family. In certain embodiments, glycoengineered CD44 glycosylated product (e.g. HCELL) is stable for at least 48 hours at 4° C., with retained expression after cell cryopreservation.

What is claimed is: 
     
         1 . A method of detecting a change of expression in cell-surface Type 2 terminal lactosamines on a population of cultured cells comprising the steps of:
 (a) contacting the cells with a glycosyltransferase and a donor nucleotide sugar, wherein the glycosyltransferase and donor nucleotide sugar are effective to enforce expression of a glycan; and   (b) detecting the product glycan on the cells,   wherein the contacting of step (a) is performed before and/or after any culture condition modification.   
     
     
         2 . The method of  claim 1 , wherein the detecting of step (b) comprises an antibody-based technique that recognizes the product glycan. 
     
     
         3 . The method of  claim 1 , wherein the detecting of step (b) is effective to precisely identify the Type 2 terminal lactosamine target of an α(1,3)-fucosyltransferase by detecting one or more of product glycans consisting of sLeX, LeX, VIM-2, and Difucosyl sLeX. 
     
     
         4 . A method of detecting differences in level of expression of cell-surface Type 2 terminal lactosamines on a population of cultured cells propagated under different conditions comprising the steps of:
 (a) culturing a first population of cells under a first culture condition;   (b) culturing a second population of cells under a second (different) culture condition;   (c) contacting the first and second population of cells with a glycosyltransferase and a donor nucleotide sugar, wherein the glycosyltransferase and donor nucleotide sugar are effective to enforce expression of a glycan; and   (d) detecting the glycan on the first and second population of cells.   
     
     
         5 . The method of  claim 4 , wherein the glycosyltransferase is an α(1,3)-fucosyltransferase, and the detection step (d) is effective to precisely identify the Type 2 terminal lactosamine target of the fucosyltransferase by detecting one or more of glycans consisting of sLeX, LeX, VIM-2, and Difucosyl sLeX. 
     
     
         6 . The method of  claim 4 , wherein the first and second culture conditions comprise different supplements. 
     
     
         7 . The method of  claim 4 , wherein the first and second population of cells are frozen and then thawed after the contacting with the glycosyltransferase of step (c). 
     
     
         8 . The method of  claim 4 , further comprising the step of (e) selecting the culture condition that is effective to produce a desired amount of Type 2 lactosaminyl glycan. 
     
     
         9 . The method of  claim 4 , wherein the glycosyltransferase is α(1,3)-fucosyltransferase VI, α(1,3)-fucosyltransferase VII, or a combination thereof. 
     
     
         10 . A process for producing GMP-grade exofucosylated cells comprising:
 (a) providing cells with a culture medium comprising a supplement, wherein the cells comprise cell surface CD44 and the supplement is effective to maintain or increase the amount of a CD44 glycoform comprising sialylated Type 2 lactosamines;   (b) expanding the cells in the culture medium; and   (c) contacting the cells with a glycosyltransferase and a donor nucleotide sugar that are effective to enforce expression of the HCELL glycoform of CD44 on the cells.   
     
     
         11 . The process of  claim 10 , further comprising the step of storing the cells at 4° C. or less;
 wherein an HCELL glycoform of CD44 is stably expressed and cell viability is maintained for at least 48 hours. 
 
     
     
         12 . The process of  claim 10 , further comprising (d) freezing and then thawing the cells, wherein the cells stably express the HCELL glycoform of CD44 after thawing. 
     
     
         13 . The process of  claim 10 , wherein the glycotransferase is selected from the group consisting of α(1,3)-fucosyltransferase III, IV, V, VI, VII, IX or a combination thereof. 
     
     
         14 . The system of  claim 13 , wherein the glycosyltransferase is effective to enforce the HCELL glycoform of CD44 on the cells. 
     
     
         15 . The system of  claim 13 , wherein the cells are human mesenchymal stem cells (hMSCs). 
     
     
         16 . The system of  claim 10 , wherein the supplement is human platelet lysate (HPL). 
     
     
         17 . The system of  claim 14 , where the cells stably express the HCELL glycoform of CD44 on the hMSCs after freezing and then thawing the cells. 
     
     
         18 . The method of  claim 6 , further comprising the step of selecting a media supplement that is effective to maintain or increase the amount of a sialylated Type 2 lactosamine on a cell.